Showing papers in "Acta Crystallographica Section D-biological Crystallography in 1997"

Refinement of macromolecular structures by the maximum-likelihood method.

Abstract: This paper reviews the mathematical basis of maximum likelihood The likelihood function for macromolecular structures is extended to include prior phase information and experimental standard uncertainties The assumption that different parts of a structure might have different errors is considered A method for estimating σA using `free' reflections is described and its effects analysed The derived equations have been implemented in the program REFMAC This has been tested on several proteins at different stages of refinement (bacterial α-amylase, cytochrome c′, cross-linked insulin and oligopeptide binding protein) The results derived using the maximum-likelihood residual are consistently better than those obtained from least-squares refinement

wARP: improvement and extension of crystallographic phases by weighted averaging of multiple-refined dummy atomic models.

Abstract: wARP is a procedure that substantially improves crystallographic phases (and subsequently electron-density maps) as an additional step after density-modification methods such as solvent flattening and averaging. The initial phase set is used to create a number of dummy atom models which are subjected to least-squares or maximum-likelihood refinement and iterative model updating in an automated refinement procedure (ARP). Averaging of the phase sets calculated from the refined output models and weighting of structure factors by their similarity to an average vector results in a phase set that improves and extends the initial phases substantially. An important requirement is that the native data have a maximum resolution beyond \sim2.4 A. The wARP procedure shortens the time-consuming step of model building in crystallographic structure determination and helps to prevent the introduction of errors.

C—H⋯O Hydrogen Bonds in β‐sheets

Abstract: A detailed analysis of the occurrence of the C-H...O hydrogen bonds in sheet regions of proteins has been presented. 11 unique protein structures with resolution 1.3 A containing beta-sheets show a widespread presence of C-H...O hydrogen bonds. These have average C(alpha).O, CH...O distances and a C(alpha)-H...O angle of 3.29, 2.38 A and 143 degrees, respectively. As in the case of N-H...O hydrogen bonds, parallel and antiparallel beta-sheet regions show the same hydrogen-bond geometry. An inverse correlation is observed between the hydrogen-bond geometries involving the C(alpha)(i)-H...O=C and the N(i+1)-H...O=C suggesting that C-H...O hydrogen bonds may act as an additional stabilizing factor. The propensity of different amino-acid residues to form such hydrogen bonds varies and shows a clear preference for valine and threonine. C-H...O hydrogen bonds involving side chains also occur extensively in beta-sheet regions.

Structure determination of minute virus of mice.

Abstract: The three-dimensional crystal structure of the single-stranded DNA-containing (`full') parvovirus, minute virus of mice (MVM), has been determined to 3.5 A resolution. Both full and empty particles of MVM were crystallized in the monoclinic space group C2 with cell dimensions of a = 448.7, b = 416.7, c = 305.3 A and β = 95.8°. Diffraction data were collected at the Cornell High Energy Synchrotron Source using an oscillation camera. The crystals have a pseudo higher R32 space group in which the particles are situated at two special positions with 32 point symmetry, separated by ½c in the hexagonal setting. The self-rotation function showed that the particles are rotated with respect to each other by 60° around the pseudo threefold axis. Subsequently, a more detailed analysis of the structure amplitudes demonstrated that the correct space-group symmetry is C2 as given above. Only one of the three twofold axes perpendicular to the threefold axis in the pseudo R32 space group is a `true' crystallographic twofold axis; the other two are only `local' non-crystallographic symmetry axes. The known canine parvovirus (CPV) structure was used as a phasing model to initiate real-space electron-density averaging phase improvement. The electron density was easily interpretable and clearly showed the amino-acid differences between MVM and CPV, although the final overall correlation coefficient was only 0.63. The structure of MVM has a large amount of icosahedrally ordered DNA, amounting to 22% of the viral genome, which is significantly more than that found in CPV.

Template convolution to enhance or detect structural features in macromolecular electron-density maps.

Abstract: A conceptually simple real-space convolution method has been developed which can be used to detect or enhance structural features in experimental macromolecular electron-density maps. The method has been implemented in a computer program (ESSENS). One application of the method is in selectively visualizing secondary-structure elements in multiple isomorphous replacement (MIR) maps of proteins, prior to map interpretation. This application is demonstrated for MIR maps of P2 myelin protein [Jones, Bergfors, Sedzik & Unge (1988). EMBO J. 7, 1597-1604; Cowan, Newcomer & Jones (1993). J. Mol. Biol. 230, 1225-1246] and glyoxalase I [Cameron, Olin, Ridderstrom, Mannervik & Jones (1997). In preparation]. Another application is in finding the optimal orientation and position of a known structural fragment (e.g. a protein domain or a ligand) in any type of electron-density map (real-space or phased molecular replacement). This application is demonstrated for the complex of acetylcholinesterase and the snake toxin fasciculin II [Harel, Kleywegt, Ravelli, Silman & Sussman (1995). Structure, 3, 1355-1366] where the toxin was located in a map phased using the molecular-replacement solution for the acetylcholinesterase alone.

Comparison of the structures of the cubic and tetragonal forms of horse-spleen apoferritin.

Abstract: Horse-spleen apoferritin is known to crystallize in three different space groups, cubic F432, tetragonal P4212 and orthorhombic P21212. A structure comparison of the cubic and tetragonal forms is presented here. Both crystal forms were obtained by the vapor-diffusion technique and data were collected at 2.26 A (cubic crystal) and 2.60 A (tetragonal crystal) resolution. Two main differences were observed between these crystal structures: (i) whereas intermolecular contacts only involve salt-bridge type interactions via cadmium ions in the cubic structure, two types of interactions are observed in the tetragonal crystal (cadmium-ion-mediated salt bridges and hydrogen-bonding interactions) and (ii) cadmium ions bound in the threefold axes of ferritin molecules exhibit lower site-occupation factors in the tetragonal structure than in the cubic one.

Bayesian correlated MAD phasing.

Abstract: A Bayesian treatment for phase calculation in the multiwavelength anomalous diffraction (MAD) technique is presented. This approach explicitly treats effects of errors correlated among measurements at different wavelengths and between Bijvoet pairs. The resulting method, which is called Bayesian correlated MAD phasing, gives proper statistical consideration to all data and does not give special treatment to data from a particular wavelength. Results obtained using Bayesian correlated MAD phasing and two other strategies on both a model test case and on data obtained in two actual MAD experiments are compared. Although all procedures performed well when the completeness of the data was high, it is shown that Bayesian correlated MAD phasing is more robust with respect to incompleteness of data than the other methods are. At 60% completeness the improvement over other methods for the examples given was nearly 50% in the correlation coefficients, and made a substantial difference in the interpretability of an electron-density map.

Bias Reduction in Phase Refinement by Modified Interference Functions: Introducing the γ Correction

Abstract: The chemical, physical and symmetry constraints of an electron-density map impose relationships between structure factors, and these relationships are exploited during refinement. However, constraints often allow an artificially high correlation between the model and the original structure factors, a flaw known as model or refinement bias. Elimination of the bias component of a constrained model, the component insensitive to constraints, enhances the power of phase-refinement techniques. The scale of the bias component, here denoted as γ, is shown to be equal in magnitude to the origin vector of the interference function G that defines the relationships between the structure factors. The γ correction leads to solvent flipping in the case of phase improvement by solvent flattening, and other types of constraint allow a similar treatment.

Structures of a blue-copper nitrite reductase and its substrate-bound complex.

Abstract: Copper-containing nitrite reductases (NiR's) have been conveniently subdivided into blue and green NiR's which are thought to be redox partners of azurins and pseudo-azurins, respectively. Crystal structures of two green NiR's have recently been determined. Alcaligenes xylosoxidans has been shown to have a blue-copper nitrite reductase (AxNiR) and two azurins with 67% homology both of which donate electrons to it effectively. The first crystal structure of a blue NiR (AxNiR) in its oxidized and nitrite-bound forms, with particular emphasis to the Cu sites, is presented. The Cu-Smet distance is the same as those in the green NiR's. Thus, the length of this interaction is unlikely to be responsible for differences in colour. Crystallographic data presented here taken together with structural data of other single Cu type-1 proteins and their mutants suggest that the displacement of Cu from the strong ligand plane is perhaps the cause for the differences in colour observed for otherwise `classical' blue Cu centre. Nitrite is observed binding to the catalytic Cu in a bidentate fashion displacing the water molecule, offering a neat rationalization for the XAFS observation that the type-2 Cu-ligand distances increase on nitrite binding as a result of increased coordination. These results are discussed in terms of enzyme mechanism.

Comparative analysis of thaumatin crystals grown on earth and in microgravity.

Abstract: The protein thaumatin was studied as a model macromolecule for crystallization in microgravity-environment experiments conducted on two US Space Shuttle missions (USML-2 and LMS). In this investigation, we have evaluated and compared the quality of space- and earth-grown thaumatin crystals using X-ray diffraction analyses, and characterized them according to crystal size, diffraction resolution limit and mosaicity. Two different approaches for growing thaumatin crystals in the microgravity environment, dialysis and liquid–liquid diffusion, were employed as a joint experiment by our two investigative teams. Thaumatin crystals grown in a microgravity environment were generally larger in volume and the total number of crystals was less, relative to crystals grown on earth. They diffracted to significantly higher resolution and with improved diffraction properties, as judged by relative plots of I/σ versus resolution. The mosaicity of space-grown crystals was significantly less than that of crystals grown on earth. Increased concentrations of protein in the crystallization chambers in microgravity led to larger crystals. The data presented here lend further support to the idea that protein crystals of improved quality can be obtained in a microgravity environment.

Structure of rabbit muscle phosphoglucomutase refined at 2.4 A resolution.

Abstract: Data between 6.0 and 2.4 A resolution, collected at 253 K, wer used to refine a revised atomic model of muscle phosphoglucomutase: final crystallographic R factor = 16.3% (Rfree = 19.1%); final r.m.s. deviations from ideal bond lengths and angles = 0.018 A and 3.2 degrees, respectively. Features of the protein that were recognized only in the revised model include: the disposition of water molecules within domain-domain interfaces; two ion pairs buried in domain-domain interfaces, one of which is a structural arginine around which the active-site phosphoserine loop is wound; the basic architecture of the active-site 'crevice', which is a groove in a 1(1/3)-turn helix, open at both ends, that is produced by the interfacing of the four domains; the distorted hexacoordinate ligand sphere of the active-site Mg2+, where the enzymic phosphate group acts as a bidentate ligand; a pair of arginine residues in domain IV that form part of the enzymic phosphate-binding site (distal subsite) whose disposition in the two monomers of the asymmetric unit is affected unequally by distant crystallographic contacts; structural differences throughout domain IV, produced by these differing contacts, that may mimic solution differences induced by substrate binding; large differences in individually refined Debye-Waller thermal factors for corresponding main-chain atoms in monomers (1) and (2), suggesting a dynamic disorder within the crystal that may involve domain-size groups of residues; and a 'nucleophilic elbow' in the active site that resides in a topological environment differing from previous descriptions of this type of structure in other proteins.

Lysozyme aggregation studied by light scattering. I. Influence of concentration and nature of electrolytes.

Abstract: Static and dynamic light scattering have been employed to investigate the behaviour of lysozyme solutions when varying the concentration of (NH(4))(2)SO(4) and NaCl for screening the repulsive forces between the monomers. At the initial aggregation stages clusters, which can be classified as mass-fractals undergoing diffusion limited-like aggregation, coexist with monomers or small lysozyme oligomers. The kinetics of fractal growth deliver observables that exhibit distinct tendencies when examined as a function of the concentration and nature of the electrolyte. The behaviour of the observables changes drastically above 0.84 M (NH(4))(2)SO(4) and 0.60 M NaCl. Static light scattering revealed a progressive restructuring of the fractals to compact structures at the latter stages of the reaction. Based on the correlations between the various observables an attempt is made to predict the long-term fate of the nucleating solutions.

X-ray structure of a ribonuclease A-uridine vanadate complex at 1.3 A resolution.

Abstract: The X-ray crystal structure of a uridine vanadate-ribonuclease A complex has been determined at 1.3 A resolution. The resulting structure includes all 124 amino-acid residues, a uridine vanadate, 131 water molecules, and a single bound 2-methyl-2-propanol. Side chains of 11 surface residues showing discrete disorder were modeled with multiple conformations. The final crystallographic R factor is 0.197. Structures obtained from high-level ab initio quantum calculations of model anionic oxyvanadate compounds were used to probe the effects of starting structure on the refinement process and final structure of the penta-coordinate phosphorane analog, uridine vanadate. The least-squares refinement procedure gave rise to the same final structure of the inhibitor despite significantly different starting models. Comparison with the previously determined complex of ribonuclease A with uridine vanadate obtained from a joint X-ray/neutron analysis (6RSA) [Wlodawer, Miller & Sjolin (1983). Proc. Natl Acad. Sci. USA, 80, 3628-3631] reveals similarities in the overall enzyme structure and the relative position of the key active-site residues, Hisl2, His119 and Lys41, but significant differences in the V-O bond distances and angles. The influence of ligand binding on the enzyme structure is assessed by a comparison of the current X-ray structure with the phosphate-free ribonuclease A structure (7RSA) [Wlodawer, Svensson, Sjolin & Gilliland (1988). Biochemistry, 27, 2705-2717]. Ligand binding alters the solvent structure, distribution and number of residues with multiple conformations, and temperature factors of the protein atoms. In fact, the temperature factors of atoms of several residues that interact with the ligand are reduced, but those of the atoms of several residues remote from the active site exhibit marked increases.

Growth Mechanism of the (110) Face of Tetragonal Lysozyme Crystals

Abstract: The measured macroscopic growth rates of the (110) face of tetragonal lysozyme show an unexpectedly complex dependence on the supersaturation. In earlier studies it has been shown that an aggregate growth unit could account for experimental growth-rate trends. In particular molecular packing and interactions in the growth of the crystal were favored by completion of the helices along the 4, axes. In this study the molecular orientations of the possible growth units and the molecular growth mechanism were identified. This indicated that growth was a two-step process: aggregate growth units corresponding to the 4, helix are first formed in the bulk solution by stronger intermolecular bonds and then attached to the crystal face by weaker bonds. A more comprehensive analysis of the measured (110) growth rates was also undertaken. They were compared with the predicted growth rates from several dislocation and two-dimensional nucleation growth models, employing tetramer and Octamer growth units in polydisperse solutions and monomer units in monodisperse solutions. The calculations consistently showed that the measured growth rates followed the expected model relations with octamer growth units, in agreement with the predictions from the molecular level analyses.

Different Tools to Study Interaction Potentials in γ‐Crystallin Solutions: Relevance to Crystal Growth

Abstract: Osmotic pressure, small-angle X-ray scattering and quasi-elastic light scattering were used to study the medium-range interaction potentials between macromolecules in solution. These potentials determine macromolecular crystallization. Calf eye lens γ-crystallins were used as a model system with the charge, and therefore the interactions, varied with pH. The second virial coefficient was determined under the same conditions with each of the three techniques. Osmotic pressure and quasi-elastic light scattering can be used conveniently in the laboratory to rapidly test the type of interactions (either attractive or repulsive) present in the solution. The measurement is direct with osmotic pressure, whereas with quasi-elastic light scattering, the directly measured coefficient is a combination of thermodynamic and hydrodynamic terms. X-rays, which require more sophisticated equipment such as synchrotron radiation facilities, can provide more detailed information on the interparticle potentials when models are used. At low ionic strength, two potentials were found necessary to account for the temperature and pH phase diagram as a function of protein concentration. The first potential is the van der Waals attractive potential that was previously shown to account for the fluid-fluid phase separation at low temperature. The second potential is an electrostatic coulombic repulsive potential which is a function of the protein charge and thus of the pH. The interaction trail could be followed at protein concentrations as low as 10 mg ml−1. The results as a whole are expected to be valid for all compact low molecular weight proteins at low ionic strength.

Comparison of ternary complexes of Pneumocystis carinii and wild-type human dihydrofolate reductase with coenzyme NADPH and a novel classical antitumor furo[2,3-d]pyrimidine antifolate.

Abstract: The novel furopyrimidine N-(4-{N-[(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)methyl]methylamino}benzoyl)-L- glutamate (MTXO), a classical antifolate with antitumor activity comparable to that of methotrexate (MTX), has been studied as inhibitor-cofactor ternary crystal complexes with wild-type Pneumocystis carinii (pc) and recombinant human wild-type dihydrofolate reductase (hDHFR). These structural data provide the first direct comparison of the binding interactions of the same antifolate inhibitor in the active site for pc and human DHFR. The human ternary DHFR complex crystallizes in the rhombohedral space group R3 and is isomorphous to the ternary complex reported for a gamma-tetrazole methotrexate analogue, MTXT. The pcDHFR complex crystallizes in the monoclinic space group P2(1) and is isomorphous to that reported for a trimethoprim (TMP) complex. Interpretation of difference Fourier electron-density maps for these ternary complexes revealed that MTXO binds with its 2,4-diaminofuropyrimidine ring interacting with Glu32 in pc and Glu30 in human DHFR, as observed for MTXT. The presence of the 6-5 furopyrimidine ring instead of the 6-6 pteridine ring results in a different bridge conformation compared with that of MTXT. The bridge torsion angles for MTXO, i.e. C(4a)-C(5)-C(8)-N(9) and C(5)-C(8)-N(9)-C(1'), are -156.5/51.9 degrees and -162.6/51.8 degrees, respectively for h and pc, compared with -146.8/57.4 degrees for MTXT. In each case, the p-aminobenzoylglutamate conformation is similar to that observed for MTXT. In the pcDHFR complex, the active-site region is conserved and the additional 20 residues in the sequence compared with the human enzyme are located in external loop regions. There is a significant change in the nicotinamide ribose conformation of the cofactor which places the nicotinamide O atom close to the 4NH(2) group of MTXO (2.7 A), a shift not observed in hDHFR structures. As a consequence of this, there is a loss of a hydrogen bond between the nicotinamide carbonyl group and the backbone of Ala12 in pcDHFR. In the human ternary complexes, the cofactor NADPH is bound with a more extended conformation, and the nicotinamide O atom makes a 3.5 A contact with the 4NH(2) group of MTXO. Although the novel classical antifolate MTXO is not highly active against pcDHFR, there are correlations between its binding interactions consistent with its lower potency as an inhibitor of h and pcDHFR compared with MTX.

X-ray structure of the 154-amino-acid form of recombinant human basic fibroblast growth factor. comparison with the truncated 146-amino-acid form.

Abstract: The crystal structure of the 154-amino-acid form of human basic fibroblast growth factor (hbFGF154), probably representing the intact form of hbFGF as deduced from the open reading frame of hbFGF cDNA, was determined by X-ray crystallography and refined to a crystallographic residual of 19.0% for all data between 20.0 and 2.0 A resolution. Crystals were obtained from recombinant hbFGF154 expressed in E. coli. hbFGF154 has the same overall structure as the N-terminus truncated 146-amino-acid form. The structure has a Kunitz-type fold and is built of 12 β-strands of which six antiparallel strands form a β-sheet barrel. In the structure it was possible to locate two additional residues at the N terminus and the last three C-terminal amino-acid residues, which seem to be disordered in all but one of the reported structures of the truncated form of hbFGF. The C-terminal amino-acid residues are part of the last β-strand through the formation of a hydrogen bond between the main-chain amide group of Ala152 and the carbonyl O atom of Pro28. An apparent phosphate ion is bound within the basic region on the surface of the molecule and has as ligands the side chains of Asn35, Arg128 and Lys133 and two water molecules. A slightly different hydrogen-bonding pattern to the phosphate ion is observed as compared with the sulfate ions in the truncated forms [Eriksson, Cousens & Matthews (1993). Protein Sci. 2, 1274–1284; Zhang, Cousens, Barr & Sprang (1991). Proc. Natl Acad. Sci. USA, 88, 3446–3450]. One molecule of β-mercaptoethanol forms a disulfide bridge to Cys77.

Ab initio structure determination and refinement of a scorpion protein toxin.

Abstract: The structure of toxin II from the scorpion Androctonus australis Hector has been determined ab initio by direct methods using SnB at 0.96 A resolution. For the purpose of this structure redetermination, undertaken as a test of the minimal function and the SnB program, the identity and sequence of the protein was withheld from part of the research team. A single solution obtained from 1 619 random atom trials was clearly revealed by the bimodal distribution of the final value of the minimal function associated with each individual trial. Five peptide fragments were identified from a conservative analysis of the initial E-map, and following several refinement cycles with X-PLOR, a model was built of the complete structure. At the end of the X-PLOR refinement, the sequence was compared with the published sequence and 57 of the 64 residues had been correctly identified. Two errors in sequence resulted from side chains with similar size while the rest of the errors were a result of severe disorder or high thermal motion in the side chains. Given the amino-acid sequence, it is estimated that the initial E-map could have produced a model containing 99% of all main-chain and 81% of side-chain atoms. The structure refinement was completed with PROFFT, including the contributions of protein H atoms, and converged at a residual of 0.158 for 30 609 data with F >or= 2sigma(F) in the resolution range 8.0-0.964 A. The final model consisted of 518 non-H protein atoms (36 disordered), 407 H atoms, and 129 water molecules (43 with occupancies less than unity). This total of 647 non-H atoms represents the largest light-atom structure solved to date.

X-ray topography of tetragonal lysozyme grown by the temperature-controlled technique.

Abstract: Growth-induced defects in lysozyme crystals were observed by white-beam and monochromatic X-ray topography at the National Synchrotron Light Source (NSLS) at the Brookhaven National Laboratory (BNL). The topographic methods were non-destructive to the extent that traditional diffraction data collection could be performed to high resolution after topography. It was found that changes in growth parameters, defect concentration as detected by X-ray topography, and the diffraction quality obtainable from the crystals were all strongly correlated. In addition, crystals with fewer defects showed lower mosaicity and higher diffraction resolution as expected.

Channels in the Gramicidin S-with-Urea Structure and their Possible Relation to Transmembrane Ion Transport

Abstract: The structure of membrane-active antibiotic cyclodecapeptide gramicidin S in the crystals of its complex with urea, C(60)H(92)N(12)0(10).0.(5)[(NH(2))(2)CO].7.94H(2)0, has been investigated with three-dimensional X-ray data by the automatic sequential approximation method. The crystals are trigonal, space group P3(1)21, a = 25.80(3), c= 21.49 (2) A, M(r) = 7968, calculated density = 1.088 mg m(-3), Z = 1. Conventional R factor: R1 = 0.0943, wR2 = 0.2478 [I> 2sigma(I)]. The molecule possesses an antiparallel twisted beta-structure, with turns involving the Phe-Pro peptides. The Orn side chains extend on one side of the sheet, while the non-polar Val and Leu side chains are located on the other face. One of the Orn residues (namely Orn2) is linked by an intermolecular hydrogen bond to the O atom of Phe4 residue, the other is free. The side chains of the Phe residues have trans orientation (chi(1) approximately 180 degrees ) and those of the Val, Orn, Leu residues, except those of Orn2, have the preferential gauche orientation with the chi(1) angle close to 60. Two side chains show statistical disorder and conformation of the Pro residues is C(s)-C(beta)-exo. There is half a urea molecule and also 7.94 water molecules distributed on 13 positions for each antibiotic molecule. A partially occupied and poorly ordered alcohol molecule had been identified. The gramicidin S molecules are arranged around the 3(1) axis in the form of a left-handed double spiral forming suggestive channels. The outer hydrophobic surface of the spiral is made of uncharged side radicals while the inside surface consists of the main-chain atoms, mainly O and N, and of ornithine side chains with N atoms at the ends. By changing the Orn side-chain conformation, the inner diameter of the channels may change from 3.4 to 6.3 A. Thus, ions and particles of rather large size may pass through the channel. The possibility of the creation of the gramicidin S channels in mitochondrial membranes has been noted by some biochemists. The channel complexes are close-packed in a hexagonal arrangement in the crystal. The CI(-) ions, present in abundance in the mother solution, are not found ordered in the crystals, which may indicate the absence of the charges in the terminal N atoms of the Orn residues.

Crystallization of Chicken Egg-White Lysozyme from Ammonium Sulfate

Abstract: Chicken egg-white lysozyme was crystallized from ammonium sulfate over the pH range 4.0–7.8, with protein concentrations from 100 to 150 mg ml−1. Crystals were obtained by vapor-diffusion or batch-crystallization methods. The protein crystallized in two morphologies with an apparent morphology dependence on temperature and protein concentration. In general, tetragonal crystals could be grown by lowering the protein concentration or temperature. Increasing the temperature or protein concentration resulted in the growth of orthorhombic crystals. Representative crystals of each morphology were selected for X-ray analysis. The tetragonal crystals belonged to the P43212 space group with crystals grown at pH 4.4 having unit-cell dimensions of a = b = 78.71, c = 38.6 A and diffracting to beyond 2.0 A. The orthorhombic crystals, grown at pH 4.8, were of space group P21212 and had unit-cell dimensions of a = 30.51, b = 56.51 and c = 73.62 A.

Lysozyme Aggregation Studied by Light Scattering. II. Variations of Protein Concentration

Abstract: Static and dynamic light scattering have been employed to investigate the behaviour of nucleating lysozyme solutions in the range between 0.34 and 3.08 mM. Preselected concentrations of NaC1 and (NH(4))(2)SO(4) have been used to screen the repulsive Coulombic interactions and trigger aggregation. Initially, mass-fractals undergoing diffusion limited-like aggregation coexist with monomers or small lysozyme oligomers. The growth kinetics of the fractals deliver observables that exhibit distinct tendencies when examined as a function of lysozyme concentration. The behaviour of the observables changes drastically around 2.0 mM lysozyme. Static light scattering experiments revealed progressive restructuring or growth of compact structures at later stages of the aggregation. Based on the correlations between the observables an attempt is made to predict whether the examined solutions will crystallize or not. A tentative scheme, involving the most prominent structures observed in nucleating lysozyme solutions, is discussed.

Partial improvement of crystal quality for microgravity-grown apocrustacyanin C1.

Abstract: The protein apocrustacyanin C1 has been crystallized by vapour diffusion in both microgravity (the NASA space shuttle USML-2 mission) and on the ground. Rocking width measurements were made on the crystals at the ESRF Swiss-Norwegian beamline using a high-resolution ψ-circle diffractometer from the University of Karlsruhe. Crystal perfection was then evaluated, from comparison of the reflection rocking curves from a total of five crystals (three grown in microgravity and two earth controls), and by plotting mosaicity versus reflection signal/noise. Comparison was then made with previous measurements of almost `perfect' lysozyme crystals grown aboard IML-2 and Spacehab-I and reported by Snell et al. [Snell, Weisgerber, Helliwell, Weckert, Holzer & Schroer (1995). Acta Cryst. D51, 1099–1102]. Overall, the best diffraction-quality apocrustacyanin C1 crystal was microgravity grown, but one earth-grown crystal was as good as one of the other microgravity-grown crystals. The remaining two crystals (one from microgravity and one from earth) were poorer than the other three and of fairly equal quality. Crystal movement during growth in microgravity, resulting from the use of vapour-diffusion geometry, may be the cause of not realising the `theoretical' limit of perfect protein crystal quality.

Structure of ferric soybean leghemoglobin a nicotinate at 2.3 A resolution.

Abstract: Soybean leghemoglobin a is a small (16 kDa) protein facilitating the transport of O2 to respiring N2-fixing bacteria at low free-O2 tension. The crystal structure of soybean ferric leghemoglobin a nicotinate has been refined at 2.3 A resolution. The final R factor is 15.8% for 6877 reflections between 6.0 and 2.3 A. The structure of soybean leghemoglobin a (143 residues) is closely similar to that of lupin leghemoglobin II (153 residues), the proteins having 82 identical residues when the sequences are aligned. The new structure provides support for the conclusion that the unique properties of leghemoglobin arise principally from a heme pocket considerably larger and more flexible than that of myoglobin, a strongly ruffled heme group, and a proximal histidine orientation more favourable to ligand binding.

Polyalanine reconstruction from Calpha positions using the program CALPHA can aid initial phasing of data by molecular replacement procedures.

Abstract: The Cα positions for a protein can provide a scaffold for the reconstruction of more complete models. Reconstructions can be by manual rebuilding, from geometric solutions to the constraints on main-chain torsion angles or from databases of known protein structures. The last method is usually the most convenient and reliable. This paper describes a database reconstruction program, CALPHA, and assesses its accuracy and reliability by test reconstructions of well refined structures. Typically, backbone atoms are repositioned to within 0.3 A of their original positions. This corresponds to regenerating main-chain torsion angles to within 15°. Uses of CALPHA for automating refinement procedures are discussed. In particular, the uses of Cα-only and rcconstructed polyalanine models of HIV-1 reverse transcriptase for cross-rotation and translation function searches are compared. The CALPHA polyalanine model is found to provide more selectivity for approximately correct orientations. The effect on the translation function is dependent on the resolution shell employed. It is expected that these observations will be applicable in other cases.

Computational support for the suggested contribution of C-H...O=C interactions to the stability of nucleic acid base pairs.

Abstract: Ab inito quantum chemical calculations are performed to quantify the stabilizing role of long C-H...O=C contacts in nucleic acid base pairing, which was suggested by Leonard, McAuley-Hecht, Brown & Hunter [(1995). Acta Cryst. D51, 136-139]. For the Watson-Crick adenine-uracil pair, a contribution of about 6% to the total bond energy is obtained. This weakly bonding effect is primarily a result of electrostatic attraction between the total positive charge of adenine C(2)-H and the negative end of the dipole uracil O(2)=C.

X-ray structure of turkey-egg lysozyme complex with tri-N-acetylchitotriose. Lack of binding ability at subsite A.

Abstract: The turkey-egg lysozyme (TEL) complex with tri-N-acetylchitotriose [(GlcNac)3] was co-crystallized from 1.5% TEL and 2 mM (GlcNac)3 at pH 4.2. The crystal structure was determined by molecular replacement and refined to an R value of 0.182 using 10–1.77 A data. The (GlcNac)3 molecule occupies the subsites A, B and C. At the subsites B and C, the sugar residues are bound in a similar manner to that found in the hen-egg lysozyme (HEL) complex. In contrast, the GlcNac residue at the subsite A is exposed to bulk solvent and has no contact with the protein molecule because the active residue Asp101 in HEL is replaced by Gly in TEL. A sulfate ion is bound in the vicinity of subsite B and forms hydrogen bonds with the sugar residue and the guanidino group of Arg61, assisting the binding of the sugar residue to subsite B. The active-site cleft of TEL is narrower than that of native TEL, thus attaining the best fit of the (GlcNac)3 molecule. The lack of binding ability of subsite A is discussed in relation to the catalytic properties of TEL. The result suggests that the cleavage pattern of oligosaccharide substrates in the catalytic reaction is regulated by the protein–sugar interaction at subsite A.

Structure of d(TGCGCA)2 and a comparison to other DNA hexamers.

Abstract: The X-ray crystal structure of d(TGCGCA)2 has been determined at 120 K to a resolution of 1.3 A. Hexamer duplexes, in the Z-DNA conformation, pack in an arrangement similar to the `pure spermine form' [Egli et al. (1991). Biochemistry, 30, 11388–11402] but with significantly different cell dimensions. The phosphate backbone exists in two equally populated discrete conformations at one nucleotide step, around phosphate 11. The structure contains two ordered cobalt hexammine molecules which have roles in stabilization of both the Z-DNA conformation of the duplex and in crystal packing. A comparison of d(TGCGCA)2 with other Z-DNA hexamer structures available in the Nucleic Acid Database illustrates the elusive nature of crystal packing. A review of the interactions with the metal cations Na+, Mg2+ and Co3+ reveals a relatively small proportion of phosphate binding and that close contacts between metal ions are common. A prediction of the water structure is compared with the observed pattern in the reported structure.

High-pressure krypton gas and statistical heavy-atom refinement: a successful combination of tools for macromolecular structure determination.

Abstract: The noble gas krypton is shown to bind to crystallized proteins in a similar way to xenon [Schiltz, Prange & Fourme (1994). J. Appl. Cryst. 27, 950-960]. Preliminary tests show that the major krypton binding sites are essentially identical to those of xenon. Noticeable substitution is achieved only at substantially higher pressures (above 50 x 10(5) Pa). As is the case for xenon, the protein complexes with krypton are highly isomorphous with the native structure so that these complexes can be used for phase determination in protein crystallography. Krypton is not as heavy as xenon, but its K-absorption edge is situated at a wavelength (0.86 A) that is readily accessible on synchrotron radiation sources. As a test case, X-ray diffraction data at the high-energy side of the K edge were collected on a crystal of porcine pancreatic elastase (molecular weight of 25.9 kDa) put under a krypton gas pressure of 56 x 10(5) Pa. The occupancy of the single Kr atom is approximately 0.5, giving isomorphous and anomalous scattering strengths of 15.2 and 1.9 e, respectively. This derivative could be used successfully for phase determination with the SIRAS method (single isomorphous replacement with anomalous scattering). After phase improvement by solvent flattening, the resulting electron-density map is of exceptionally high quality, and has a correlation coefficient of 0.85 with a map calculated from the refined native structure. Careful data collection and processing, as well as the correct statistical treatment of isomorphous and anomalous signals have proven to be crucial in the determination of this electron-density map. Heavy-atom refinement and phasing were carried out with the program SHARP, which is a fully fledged implementation of the maximum-likelihood theory for heavy-atom refinement [Bricogne (1991). Crystallographic Computing 5, edited by D. Moras, A. D. Podjarny & J. C. Thierry, pp. 257-297. Oxford: Clarendon Press]. It is concluded that the use of xenon and krypton derivatives, when they can be obtained, associated with statistical heavy-atom refinement will allow one to overcome the two major limitations of the isomorphous replacement method i.e. non-isomorphism and the problem of optimal estimation of heavy-atom parameters.

Crystallization and preliminary X-ray analysis of the thermostable alkaline-tolerant xylanase from Bacillus stearothermophilus T-6.

Abstract: The extracellular thermostable xylanase (XT-6) produced by the thermophilic bacterium Bacillus stearothermophilus T-6 was shown to bleach pulp optimally at pH 9 and 338 K, and was successfully used in a large-scale biobleaching mill trial. The xylanase gene was cloned and sequenced. The mature enzyme consists of 379 amino acids with a calculated molecular weight of 43 808 and pI of 9.0. Crystallographic studies of XT-6 were initiated to study the mechanism of catalysis as well as to provide a structural basis for rational introduction of enhanced thermostability by site-specific mutagenesis. This report describes the crystallization and preliminary crystallographic characterization of the native XT-6 enzyme. The most suitable crystals were obtained by the vapor-diffusion method using ammonium sulfate and 2-methyl-2,4-pentanediol as an organic additive. The crystals belong to a primitive trigonal crystal system (space group P31 or P32) with room-temperature cell dimensions of a = b = 114.9 and c = 122.6 A. At 103 K the volume of the unit cell decreased significantly with observed dimensions of a = b = 112.2 and c = 122.9 A. These crystals are mechanically strong and diffract X-rays to better than 2.2 A resolution. The crystals exhibit considerable radiation damage at room temperature even at relatively short exposures to X-rays. A full 2.3 A resolution diffraction data set (99.8% completeness) has recently been collected on flash-frozen crystals at 103 K using synchrotron radiation. Two derivatives of XT-6 were recently prepared. In the first derivative, a unique Cys residue replaced Glu265, the putative nucleophile in the active site. The second derivative was selenomethionyl xylanase which was produced biosynthetically. These derivatives have been crystallized and the resulting crystals were shown to be isomorphous to the native crystals and diffract X-rays to comparable resolutions.